Inosine monophosphate dehydrogenase (IMPDH) has been shown to be a key enzyme in the regulation of cell proliferation and differentiation. Nucleotides are required for cells to divide and replicate. In mammals, nucleotides may be synthesized through one of two pathways: the de novo synthesis pathway or the salvage pathway. The extent of utilization of each pathway is dependent on the cell type. This selectivity has ramifications with regard to therapeutic utility as described below.
IMPDH is involved in the de novo synthesis of guanosine nucleotides. IMPDH catalyzes the irreversible NAD-dependent oxidation of inosine-5′-monophosphate (“IMP”) to xanthosine-5′-monophosphate (“XMP”), Jackson et al., Nature 256:331-333 (1975).
IMPDH is ubiquitous in eukaryotes, bacteria and protozoa. The prokaryotic forms share 30-40% sequence identity with the human enzyme.
Two distinct cDNA's encoding IMPDH have been identified and isolated. These transcripts are labeled type I and type II and are of identical size (514 amino acids). Collart et al., J. Biol. Chem. 263:15769-15772 (1988); Natsumeda et al., J. Biol. Chem. 265:5292-5295 (1990); and U.S. Pat. No. 5,665,583 to Collart et al. These isoforms share 84% sequence identity. IMPDH type I and type II form tetramers in solution, the enzymatically active unit.
B and T-lymphocytes depend on the de novo, rather than salvage pathway, to generate sufficient levels of nucleotides necessary to initiate a proliferative response to mitogen or antigen. Due to the B and T cell's unique reliance on the de novo pathway, IMPDH is an attractive target for selectively inhibiting the immune system without also inhibiting the proliferation of other cells.
Immunosuppression has been achieved by inhibiting a variety of enzymes. Examples include: phosphatase calcineurin (inhibited by cyclosporin and FK-506); dihydroorotate dehydrogenase (DHODase), an enzyme involved in the biosynthesis of pyrimidines (inhibited by leflunomide and brequinar); the kinase FRAP (inhibited by rapamycin); and the heat shock protein hsp70 (inhibited by deoxyspergualin).
Inhibitors of IMPDH have also been described in the art. WO 97/40028 and U.S. Pat. No. 5,807,876 describe a class of urea derivatives that possess a common urea backbone. WO 98/40381 describes a series of heterocyclic substituted anilines as inhibitors of IMPDH.
U.S. Pat. Nos. 5,380,879 and 5,444,072 and PCT publications WO 94/01105 and WO 94/12184 describe mycophenolic acid (“MPA”) and some of its derivatives as potent, uncompetitive, reversible inhibitors of human IMPDH type I and type II. MPA has been demonstrated to block the response of B and T-cells to mitogen or antigen. Immunosuppressants, such as MPA and derivatives of MPA, are useful drugs in the treatment of transplant rejection and autoimmune disorders, psoriasis, inflammatory diseases, including, rheumatoid arthritis, tumors and for the treatment of allograft rejection. These are described in U.S. Pat. Nos. 4,686,234, 4,725,622, 4,727,069, 4,753,935, 4,786,637, 4,808,592, 4,861,776, 4,868,153, 4,948,793, 4,952,579, 4,959,387, 4,992,467, and 5,247,083.
The combination of agents for prevention and/or treatment of IMPDH-associated disorders, especially allograft rejection, has been investigated. In one study, it was observed that cyclic AMP agonists, such as Rolipram (Schering AG), a Type 4 Phosphodiesterase Inhibitor (PDE4) and immunomodulator, synergized with IMPDH inhibitor MPA by a cAMP- and IMPDH-dependent dependent mechanism. (P. A. Canelos, L. M. Lichtenstein, S. K. Huang, D. M. Essayan, J. Allergy and Clinical Immunology, 107:593 (2001)). The chemical structure of Rolipram is [4-[3-(cyclopentyloxy)-4-methoxy-phenyl]-2-pyrrolidinone]. The investigators found that cyclic AMP agonists, such as the PDE4 inhibitor Rolipram (Rol), markedly downregulated antigen-specific T lymphocyte responses through their effects on a variety of signaling pathways. In the study, the potential for a very low concentration of Rol (10−7 M, approximate IC10) to synergize with a variety of immunosuppressive agents used for the prevention and/or treatment of allograft rejection was defined. While little or no synergistic effect on inhibition of antigen-induced proliferation (assessed by 3H Thymidine incorporation) could be demonstrated with calcineurin antagonists (cyclosporine and tacrolimus), sirolimus, or corticosteroids, a marked synergistic effect was demonstrated with MPA, the active metabolite of mycophenolate mofetil (CellCept, Roche). This effect was statistically significant over 4 orders of magnitude (10−6 to 10−9 M). This synergism was recapitulated with dibuteryl-cAMP (2×10−6 M, approximate IC10) and inhibited with the use of H-9, suggesting a mechanism involving both cAMP and protein kinase A.
Since MPA is a selective, uncompetitive, and reversible inhibitor of IMPDH, a key enzyme in the purine salvage pathway, the potential for cAMP-mediated cross-talk at this locus was further investigated. It was found that gene expression for IMPDH types I and II (assessed by RT-PCR) remained unaffected by the administration of rolipram, MPA, or both at low and high concentrations. However, functional reversal of the synergistic effect was demonstrated with the use of deoxyguanosine, a specific antagonist of MPA on IMPDH (% inhibition of proliferation 81±16 vs. 35±12, p<0.05). Finally, despite a marked synergistic effect on inhibition of proliferation, no significant downregulation in the generation of proinflammatory cytokines (IL-2, IL-4, and IFN, each assessed by RT-PCR), could be detected with the administration of Rol 10−7 M, MPA 10−8 M, or the combination. It was concluded that Rol demonstrates marked synergy with MPA by a cAMP-and IMPDH-dependent mechanism. The utility of this combination of agents for the induction of T cell tolerance was suggested by the specificity of the observed effect for proliferation, without the abrogation of cytokine generation and early signaling processes.
Tiazofurin, ribavirin and mizoribine also inhibit IMPDH. These nucleoside analogs are competitive inhibitors of IMPDH; however, these agents inhibit other NAD dependent enzymes. This low level of selectivity for IMPDH limits the therapeutic application of tiazofurin, ribavirin and mizoribine. Thus, new agents which have improved selectivity for IMPDH would represent a significant improvement over the nucleoside analogs.
Mycophenolate mofetil, sold under the trade name CELLCEPT, is a prodrug which liberates MPA in vivo. It is approved for use in preventing acute renal allograft rejection following kidney transplantation. The side effect profile limits the therapeutic potential of this drug. MPA is rapidly metabolized to the inactive glucuronide in vivo. In humans, the blood levels of glucuronide exceed that of MPA. The glucuronide undergoes enterohepatic recycling causing accumulation of MPA in the bile and subsequently in the gastrointestinal w tract. This together with the production of the inactive glucuronide effectively lowers the drug's in vivo potency, while increasing its undesirable gastrointestinal side effects.
Unlike type I, type II mRNA is preferentially upregulated in human leukemic cell lines K562 and HL-60. Weber, J. Biol. Chem. 266: 506-509 (1991). In addition, cells from human ovarian tumors and leukemic cells from patients with chronic granulocytic, lymphocytic and acute myeloid leukemias also display an up regulation type II mRNA. This disproportionate increase in IMPDH activity in malignant cells may be addressed through the use of an appropriate IMPDH inhibitor. IMPDH has also been shown to play a role in the proliferation of smooth muscle cells, indicating that inhibitors of IMPDH may be useful in preventing restenosis or other hyperproliferative vascular diseases.
IMPDH has been shown to play a role in viral replication in some viral cell lines. Carr, J. Biol. Chem. 268:27286-27290 (1993). The IMPDH inhibitor VX-497, is currently being evaluated for the treatment of hepatitis C virus in humans. Ribavirin has also been used in the treatment of hepatitis C and B viruses and when used in combination with interferon an enhancement in activity was observed. The IMPDH inhibitor ribavirin is limited by its lack of a sustained response in monotherapy and broad cellular toxicity.
There remains a need for potent selective inhibitors of IMPDH with improved pharmacological properties, physical properties and fewer side effects. Such inhibitors would have therapeutic potential as immunosuppressants, anti-cancer agents, anti-vascular hyperproliferative agents, antiinflammatory agents, antifungal agents, antipsoriatic and anti-viral agents. The compounds of the present invention are effective inhibitors of IMPDH.